JP3909008B2 - Keratinase inhibitor and skin external preparation containing the same - Google Patents

Description

【００３０】
＜実施例２＞
下記に示す処方に従って、本発明の皮膚外用剤である化粧料（ローション）を作成した。即ち、処方成分を室温で可溶化し、濾過してローションを得た。同時に角化酵素抑制剤を水に置換した比較例１のローションも同様に作成した。これらを用いて、ピーリングタイプの脱毛剤の前処置を行った。即ち、専門パネラーを用いて、これらのローションで１日１回脱毛予定部位（脛部）を７日間処置し、ピーリングタイプの脱毛剤で脱毛し、その容易さを官能検査により評価した。評価は、スコア５：極めて容易、スコア４：容易、スコア３：容易、スコア２：やや困難、スコア１：困難の基準で、スコア付けを行った。表にスコア値を示す。これより、本発明の皮膚外用剤の前処置を行った群は何れも比較例１投与群よりも色差が小さく、脱毛が容易であったことがわかる。
表２に記載の成分 ０．１重量部
１，２−ヘキシレングリコール ２ 重量部
イソプレングリコール ２ 重量部
グリセリン ２ 重量部
１，３−ブタンジオール ２ 重量部
フェノキシエタノール ０．５重量部
エタノール ５ 重量部
ＰＯＥ（６０）硬化ヒマシ油 ０．１重量部
水 ８６．３重量部
【００３１】
【表２】

【００３２】
＜実施例８＞
以下に示す処方に従って、本発明の皮膚外用剤である抗真菌剤の前処置剤を作成した。このものを用いて、モルモットフットパッドモデルでの感染実験を行った。即ち、ハートレー系白色種モルモット１群５匹のフットパッドに、マイクロスポリジウム・メンタグロファイデスの分生子を１０^６個／ｍｌ含有するリン酸緩衝生理食塩水液０．１ｍｌを２４時間クローズドパッチし感染させ、１週間後に治療実験を開始した。治療は１群は本発明の前処置剤で処置した後に後記の抗真菌ローションを塗布した。もう１群は前処置剤の角化酵素抑制剤１を水に置換した比較例２のローションで同様の前処置を行い、抗真菌ローションで治療した。治療は３週間行い、治療終了後フットパッドから皮膚片を採取し、サブロー改変培地に１０個置き、１週間培養し、真菌の生えてきたものを陽性とし、陽性率を求め、群の平均陽性率を算出した。結果を表３に示す。これより、本発明の皮膚外用剤で前処置した群の方が陽性率が低く、薬剤の浸透性が良好であったことがわかる。
（前処置用の皮膚外用剤）
角化酵素抑制剤１ １ 重量部
エタノール ４９ 重量部
ＰＯＰ・ＰＯＥコポリマー ３ 重量部
水 ４７ 重量部
（抗真菌ローション）
テルビナフィン ２ 重量部
エタノール ５０ 重量部
エチルセルロース ２ 重量部
１，３−ブタンジオール ６ 重量部
水 ４０ 重量部
【００３３】
【表３】

【００３４】
【発明の効果】
本発明によれば、皮膚乃至はその周辺組織の処置に於いて、角化酵素を阻害する手段を提供することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an external preparation for skin, and more particularly to an external preparation for skin suitable for suppressing keratin formation.
[0002]
[Prior art]
When considering the function of the skin or its surrounding tissues, keratin formation is one of the major themes. The stratum corneum has a protective function from the outside of the living body, and at the same time functions to prevent a locally administered drug from reaching the affected area for a transcutaneous treatment or the like. In hair, promoting keratinization is also important from the viewpoint of hair growth or hair restoration in order to form hair with firm hair that is difficult to come off. It is considered preferable to suppress the formation because the damage to the living body is reduced. That is, it can be said that controlling keratin formation, in other words, keratinization, is an important technique in the treatment of the skin or surrounding tissues.
[0003]
In keratinization, it is already known that keratinase plays an important role, and as an action against keratinase, estradiol derivatives (Japanese Patent Application Laid-Open No. Hei. 8-20523), herbal medicine extracts such as aloe, rosemary, carrot, ginger, tokyo, etc. (JP-A-8-20522), piroctoolamine (JP-A-8-20521), etc. are known. unknown. That is, development of means for suppressing keratinase is desired.
[0004]
In terms of hair removal, N- (carboxymethyl) phenylalanyl-β-alanine (JP 2001-172197), malonic acid amide (JP 2000-344614), carboxyalkylamines are used as hair growth inhibitors by inhibiting elastase. (JP 2000-178163), hydroxamic acids (JP 2000-169324), extract of Ichihatsu, Onis, soybean, kuzu, scented squirrel, higi, Hitosumidae, hop, etc. (JP 11-322548), sanjiko, turmeric Herbal medicines such as citrus, shy, juniper, turkey, and apricot (Japanese Patent Laid-Open No. 10-139639), scallop, gobaishi, chotsutsushi, broom fushi, jiou, bankouka, cuckoo, sakuhakuhi, nyuko, shouma, sandalwood, kika, daiou Extracts (JP-A-8-81336) and the like are known, but none of them is known to be related to keratinase.
[0005]
From the viewpoint of keratin softening or dissolution, as a keratin softening agent or solubilizer, fermented milk extract (JP 2002-241289), sulfur-containing compounds such as betaine and carbon disulfide (JP 2002-138033), papain, trypsin, etc. Denatured proteins (Japanese Patent Laid-Open No. 2000-273182) are known, but none of these are known to be related to keratinase.
[0006]
As the action on the skin of plants of the family Bambooaceae, tyrosinase inhibitory action (Japanese Patent Laid-Open No. 2000-247864), tsunami promoting action (Japanese Patent Laid-Open No. 2000-103718), substance P antagonistic action (Japanese Patent Laid-Open No. Hei 10-158179) are known. However, the relationship with keratinase is not known at all.
[0007]
[Problems to be solved by the invention]
The present invention has been made under such circumstances, and provides a means for inhibiting keratinase in order to control keratin formation, in other words, keratinization, in the treatment of skin or surrounding tissues. This is the issue.
[0008]
[Means for solving problems]
In view of such a situation, the present inventors sought a means for inhibiting keratinase, and as a result of intensive research efforts, found such an action in the plant extract of the family Gramineae, The invention has been completed. That is, this invention relates to the technique shown below.
(1) A keratinase inhibitor comprising an extract of a plant belonging to the family Gramineae.
(2) The keratinase inhibitor according to (1), wherein the plant belonging to the family Gramineae is a moss mushroom, mushroom, nematode, kokuchiku or kusasakusa.
(3) The keratinase inhibitor according to (1) or (2), wherein the extract is a polar solvent extract or a solvent removed product thereof.
(4) The keratinase inhibitor according to any one of (1) to (3), wherein the extract is a leaf extract or a solvent-removed product thereof.
(5) A skin external preparation containing the keratinase inhibitor according to any one of (1) to (4).
(6) The external preparation for skin according to (5), which is used for suppressing keratin formation.
(7) The use is for pre-treatment and / or post-treatment of hair removal, pre-treatment and / or post-treatment of an antifungal agent, or pre-treatment and / or post-treatment of treatment of hyperkeratosis The external preparation for skin according to (5) or (6), which is characterized.
(8) The external preparation for skin according to any one of (5) to (7), which is a cosmetic.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
(1) Keratinase inhibitor of the present invention The keratinase inhibitor of the present invention comprises an extract of a plant belonging to the family Gramineae. As such a plant belonging to the family Gramineae, for example, plants such as Moso bamboo, Makitake, Negararidake, Kokuchiku or Kosasakusa can be preferably exemplified, and among these, Kosasakusa can be particularly suitably exemplified. Here, as an extract, the plant itself, a processed product of the plant, an extract obtained by adding a solvent to the plant or the processed product of the plant, a solvent-removed product of the extract obtained by removing the solvent from the extract, Any of the extract and the purified product obtained by fractionating and purifying the solvent-removed product can be used. Among such extracts, an extract or a solvent removed product thereof is preferable. As the extract, a solvent-removed product of the extract extracted with a highly polar solvent can be particularly preferably exemplified. Examples of highly polar solvents include ethers such as diethyl ether, isopropyl ether and tetrahydrofuran, halogenated hydrocarbons such as methylene chloride and chloroform, esters such as ethyl acetate and methyl formate, ketones such as acetone and methyl ethyl ketone, acetonitrile Preferred examples include nitriles such as 1,3-butanediol, alcohols such as ethanol and isopropyl alcohol, and water. Among these, alcohol and / or water are particularly preferable. Extraction may be performed by adding 1 to 10 times the weight of the solvent to the plant body and immersing for several days at room temperature and for several hours at a temperature near the boiling point. After extraction, it is preferable to remove the solvent by vacuum concentration or the like, if necessary. A plant part suitable for producing such an extract is not particularly limited, but leaves are particularly preferred. The extract thus obtained is one of the enzymes involved in various chemical reactions in vivo as a biocatalyst, and is widely distributed mainly in the epidermis of hair and skin, and is involved in the synthesis of keratin and other hair cuticles. Or cortex biosynthesis, or the activity of inhibiting the activity of keratinase, an enzyme that has the function of promoting the biosynthesis of the horny layer of the skin. Examples of the keratinase include sulfhydryl oxidase (SH oxidase) and transglutaminase (T-glutaminase). The keratinase inhibitor of the present invention suitably suppresses the activity of SH oxidase. In the external preparation for skin of the present invention, the keratinization inhibitor of the present invention can contain only one species, or can contain two or more species in combination. The preferable content of the keratinase inhibitor of the present invention in the external preparation for skin of the present invention is 0.01 to 10% by weight, more preferably 0.8%, based on the total amount of the external preparation for skin. 1 to 5% by weight. This is because if the amount is too small, the activity-inhibiting action of each enzyme may not be exhibited, and if it is too much, the inhibiting action reaches its peak, and the stability of the formulation may be impaired.
[0010]
<Production Example 1>
5 kg of 50% ethanol aqueous solution was added to 1 kg of crispy leaves and heated to reflux for 3 hours. After cooling, insolubles were removed by filtration, concentrated under reduced pressure, and then freeze-dried to obtain extract 1 (keratinase inhibitor 1).
[0011]
<Production Example 2>
The Kosasakusa of Production Example 1 was treated in the same manner instead of Mosouchiku to obtain Extract 2 (Keratinase Inhibitor 2).
[0012]
<Production Example 3>
The Kosasakusa of Production Example 1 was treated in the same manner instead of Makitake to obtain Extract 3 (Keratinase Inhibitor 3).
[0013]
<Production Example 4>
The extract 4 (keratinase inhibitor 4) was obtained in the same manner as in Example 1 except that the crunchy crust was replaced with the nematode mushroom.
[0014]
<Production Example 5>
The extract 5 (keratinase inhibitor 5) was obtained in the same manner as in Example 1 except that the texture was changed to the texture.
[0015]
<Production Example 6>
500 ml of 1,3-butanediol was added to 1 kg of cassava leaves, heated at 90 ° C. for 3 hours, allowed to cool, and then insolubles were removed by filtration to obtain extract 6 (keratinase inhibitor 6).
[0016]
(2) External preparation for skin of the present invention The external preparation for skin of the present invention is characterized by containing the keratinase inhibitor of the present invention. In the present invention, the external preparation for skin means a general term for compositions administered externally to the skin, and is a term encompassing cosmetics, external preparations for skin, disinfectants for external use, and the like. A particularly preferred form as a skin external preparation of the present invention is a cosmetic. This is because the action of the keratinization inhibitor of the present invention is extremely relaxed and suitable for continuous administration. The external preparation for skin of the present invention is used for the purpose of suppressing keratinization. By suppressing keratinization, it is possible to return the site with increased keratinization to a normal shape, improve drug permeability, etc., and to administer before hair removal treatment, cuticles and cortex of hair It suppresses biosynthesis, makes the hair soft, allows hair removal treatment to be performed promptly, and reduces the burden on the living body during hair removal. Further, by administering after the hair removal treatment, the hair of the hair that grows subsequently becomes soft, and the next hair removal can be performed more easily.
[0017]
In the external preparation for skin of the present invention, in addition to each enzyme inhibitor as the essential component, an optional component usually used in external preparations for skin such as cosmetics can be contained. Examples of such optional components include hydrocarbons such as squalane, liquid paraffin, light liquid isoparaffin, heavy liquid isoparaffin, microcrystalline wax, and solid paraffin, dimethicone, femethicone, cyclomethicone, amodimethicone, polyether-modified silicone, and the like. Silicones, jojoba oil, carnauba wax, mole, beeswax, geiwa, octyldodecyl oleate, isopropyl myristate, neopentyl glycol diisostearate, malate diisostearate, stearic acid, lauric acid, Fatty acids such as myristic acid, palmitic acid, isostearic acid, isopalmitic acid, behenic acid, oleic acid, behenyl alcohol, cetanol, oleyl alcohol, octadecyl alcohol High alcohols such as coal, castor oil, coconut oil, hydrogenated coconut oil, coconut oil, wheat germ oil, triglycerides such as isostearic acid triglyceride, isooctanoic acid triglyceride, olive oil, 1,3-butanediol, glycerin, diglycerin , Dipropylene glycol, polyethylene glycol, 1,2-pentanediol, 1,2-hexylene glycol, isoprene glycol and other polyhydric alcohols, sorbitan sesquioleate, sorbitan monooleate, sorbitan trioleate, sorbitan sesquistearate, sorbitan mono Stearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene stearate, polyoxyethylene oleate, polio Nonionic surfactants such as ciethylene glyceryl fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, anionic surfactants such as sodium lauryl stearate, polyoxyethylene alkyl sulfate, sulfosuccinate ester salt, Cationic surfactants such as quaternary alkyl ammonium salts, amphoteric surfactants such as alkyl betaines, organic powders such as crystalline cellulose, cross-linked methylpolysiloxane, polyethylene powder, acrylic resin powder, talc, mica, Sericite, magnesium carbonate, calcium carbonate, titanium dioxide, iron oxide, bitumen, ultramarine, titanium mica, titanium sericite, silica and other surface-treated powders, acrylic acid / alkyl methacrylate copolymers and / or Its salt, carboxybi Nyl polymers and / or salts thereof, thickeners such as xanthan gum and hydroxypropyl cellulose, vitamins such as retinol, retinoic acid, tocopherol, riboflavin, pyridoxine, ascorbic acid, ascorbic acid phosphate ester, glycyrrhizinate, glycyrrhetin, ursolic acid , Terpenes such as oleanolic acid, active ingredients such as steroids such as estradiol, ethinyl estradiol, estriol, preservatives such as phenoxyethanol, parabens, hibiten gluconate, benzalkonium chloride, dimethylaminobenzoic acid esters, cinnamon Preferred examples include ultraviolet absorbers such as acid esters and benzophenones. The external preparation for skin of the present invention can be produced by treating such essential components and optional components according to a conventional method.
[0018]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.
[0019]
<Example 1>
Examination of action of keratin enzyme inhibitor on keratinase The keratinase inhibitory action was examined using extracts 1 to 6 of Production Examples 1 to 6. That is, after removing the back of 7 groups of 5 C3H mice (male, body weight 25-35 g), the hair removal skin of 6 groups of mice was promoted to inhibit keratinase inhibition obtained in each of the above production examples. A 70% ethanol aqueous solution containing 5% by weight of the agent was added to each of the remaining groups of mice with a 70% ethanol aqueous solution as a control, 40 μL per animal, once a day, 5 days a week. The dose was administered for 2 weeks. Six hours after the final administration, the skin of the site subjected to the above treatment was collected from each group of mice, and each was added to 9 volumes of 1 mM EDTA-containing 50 mM sodium dihydrogen phosphate aqueous solution and homogenized. . The homogenate was centrifuged at 27000 G, and the keratinase activity values of the keratinase inhibitor-administered group and the control group were measured by the following method using the obtained supernatant.
[0020]
50 mmol phosphate buffer (pH 7.6, hereinafter simply referred to as buffer) containing 0.4 mL of 2 mmol of DTT (dithiothreitol) aqueous solution and 1 mmol of EDTA in 0.4 mL of the supernatant obtained above. Sample 1 was added with 7 mL. Sample 2 was prepared by adding 1.1 mL of buffer to 0.1 mL of 2 mM DTT aqueous solution.
[0021]
Immediately after preparing Sample 1, 0.3 mL is sampled and transferred to a tube containing 3 mL of buffer containing 0.17 mmol of DTNB (5,5′-dithiobis-2-nitrobenzoic acid) in advance at 412 nm. The absorbance A1 was measured. Sample 2 was treated in the same manner, and absorbance A2 was measured.
[0022]
The remaining sample 1 was incubated at 37 ° C. for 30 minutes, and then 0.3 mL thereof was sampled and transferred to a tube to which 3 mL of a buffer containing 0.17 mmol of DTNB had previously been added, and the absorbance A3 at 412 nm was measured. did. Further, the remainder of specimen 2 was incubated at 37 ° C. for 30 minutes, and then treated in the same manner to measure absorbance A4.
[0023]
From the absorbances A1 to A4 measured in this way, A5 is obtained using the following equation, and the amount of oxidized DTT is calculated using the molar extinction count (13000 / mol · cm) of A5 and DTNB. This was determined as the keratinase activity value (Ac).
[0024]
(formula)
A5 = A1-A3- (A2-A4)
[0025]
Moreover, about the supernatant obtained above, the amount of DNA of each supernatant was measured according to a conventional method using Hoechst 33258, a DNA amount measuring reagent manufactured by Hoechst, using calf breast DNA as a standard.
[0026]
The keratinase activity value (Ac) obtained by the above method was divided by the above DNA amount for each of the keratinase inhibitor administration groups of the above Examples, and the keratinase activity per unit DNA amount was evaluated. A value (AcD, average value of 5 animals) was obtained, and this was divided by the keratinase activity value (average value of 5 animals) per unit DNA amount of the control group obtained in the same manner and multiplied by 100 (AeD) was used. AcD is a value representing keratinase activity per number of cells.
[0028]
Further, with respect to the supernatant obtained above, the protein content of each supernatant was measured using a protein assay reagent kit of PIERCE, with bovine serum albumin as a standard, according to a conventional method, and each keratinase activity was measured. By dividing the value (Ac) by this, the keratinase activity value per protein (average value of 5 AcP animals) was obtained, and this was similarly determined to determine the keratinase activity value per protein of the control group (average of 5 animals). The value (AeP) obtained by dividing by 100 and multiplying this by 100 was obtained and used together with AeD for evaluation. The results are shown in Table 1. From this, it can be seen that the keratinase inhibitor of the present invention has an activity of inhibiting the activity of SH oxidase, which is one of the keratinases.
[0029]
[Table 1]

[0030]
<Example 2>
A cosmetic (lotion) that is an external preparation for skin of the present invention was prepared according to the formulation shown below. That is, the formulation components were solubilized at room temperature and filtered to obtain a lotion. At the same time, a lotion of Comparative Example 1 in which the keratinase inhibitor was replaced with water was prepared in the same manner. Using these, pretreatment of a peeling type hair removal agent was performed. That is, using a special paneler, the hair removal planned site (shin part) was treated with these lotions once a day for 7 days, and the hair was removed with a peeling type hair remover, and the ease was evaluated by a sensory test. Evaluation was scored on the basis of score 5: extremely easy, score 4: easy, score 3: easy, score 2: somewhat difficult, score 1: difficult. The score values are shown in the table. From this, it can be seen that all the groups subjected to the pretreatment with the external preparation for skin of the present invention had a smaller color difference than the group administered with Comparative Example 1, and hair removal was easy.
Ingredients listed in Table 2 0.1 parts by weight 1,2-hexylene glycol 2 parts by weight isoprene glycol 2 parts by weight glycerin 2 parts by weight 1,3-butanediol 2 parts by weight phenoxyethanol 0.5 parts by weight ethanol 5 parts by weight POE (60) hydrogenated castor oil 0.1 parts by weight water 86.3 parts by weight
[Table 2]

[0032]
<Example 8>
According to the formulation shown below, a pretreatment agent for an antifungal agent which is an external preparation for skin of the present invention was prepared. Using this, an infection experiment was conducted using a guinea pig footpad model. That is, 0.1 ml of phosphate buffered saline solution containing 10 ⁶ / ml of conidia of microsporidium mentagrophides was closed patch for 24 hours on a foot pad of 5 Hartley white guinea pigs. The treatment experiment was started one week later. In treatment, one group was treated with the pretreatment agent of the present invention, and then the antifungal lotion described below was applied. The other group was treated with an antifungal lotion by the same pretreatment with the lotion of Comparative Example 2 in which the pretreatment keratinase inhibitor 1 was replaced with water. The treatment is performed for 3 weeks. After the treatment is completed, a piece of skin is collected from the foot pad, placed in Sabouraud modified medium, cultured for 1 week, the fungus is positive, the positive rate is obtained, and the average positive of the group The rate was calculated. The results are shown in Table 3. This shows that the group pretreated with the external preparation for skin of the present invention had a lower positive rate and better drug permeability.
(External skin preparation for pretreatment)
Keratinase inhibitor 1 1 part by weight ethanol 49 parts by weight POP / POE copolymer 3 parts by weight water 47 parts by weight (antifungal lotion)
Terbinafine 2 parts by weight Ethanol 50 parts by weight Ethylcellulose 2 parts by weight 1,3-butanediol 6 parts by weight Water 40 parts by weight
[Table 3]

[0034]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the means which inhibits a keratin enzyme can be provided in the treatment of skin or its surrounding tissue.

Claims (6)

A skin external preparation, which is a cosmetic for softening hair, comprising an extract having a sulfhydryl oxidase inhibitory activity or a solvent-removed product of a plant belonging to the family Gramineae . The topical skin preparation according to claim 1, wherein the plant part from which the extract or solvent-removed product having the sulfhydryl oxidase inhibitory activity of the plant belonging to the family Gramineae is a leaf. The skin external preparation according to claim 1 or 2, wherein the plant belonging to the family Gramineae is Subtilis, Mushroom, Negaritake, Kokuchiku or Kosasakusa. A method for producing an external preparation for skin, which is a cosmetic for hair softening, comprising an extract having a sulfhydryl oxidase inhibitory activity or a solvent-removed product of a plant belonging to the family Gramineae. The production method according to claim 4, wherein the plant body part from which the extract or solvent-removed product having the sulfhydryl oxidase inhibitory activity of the plant belonging to the family Gramineae is a leaf. 6. The production method according to claim 4 or 5, wherein the plant belonging to the family Gramineae is Subtilis, Mushroom, Negaritake, Kokuchiku or Kosasakusa.